Reactive oxygen species (ROS) include superoxide and its downstream metabolites. These species are known to play multiple roles in both physiology and pathophysiology. A prominent source of ROS in vivo are the NADPH oxidase (Nox) family of enzymes that in humans consists of 7 isoforms (Nox1-5, Duox1, Duox2) with distinct tissue distribution and mechanisms of regulation. The prototypic family member (Nox2) is the classic respiratory burst oxidase' that produces high levels of ROS under strict regulation that are critical for host defence. ROS important in cellular signaling are produced at more modest levels, often by other Nox isoforms, and have been described in other cell types. In this regard, the Nox4 NADPH oxidase isoform is of particular interest as it constitutively generates ROS in the form of hydrogen peroxide (H2O2) and is regulated principally at the transcriptional level. Emerging data from our previous funding period indicate that Nox4, in contrast to O2- producing NADPH oxidase isoforms, promotes physiological vascular adaptation and tissue repair. In this application, we present data supporting our central hypothesis that endothelial Nox4 is required for the adaptive vascular effects of endurance exercise including enhanced NO bioactivity and thrombosis resistance. To investigate this hypothesis, we will first determine the in vivo role of Nox4 in the vascular response to endurance exercise. For these studies, Nox4-/- and wild-type mice will undergo endurance exercise followed by assessment of vascular adaptation determined as eNOS/NO bioactivity, and upregulation of antithrombotic (KLF2, thrombomodulin) and antioxidant (Nrf2, PGC-1) pathways. To determine the specific impact of endothelial Nox4, we will also test exercise-induced vascular adaptation in constitutive and inducible endothelial- specific Nox4 knockout (ECKONox4) models we have created and characterized. We will then determine the role of antioxidant gene regulation in the Nox4 response to endurance exercise as our preliminary data indicate that Nox4 upregulates both Nrf2- and PGC-1-dependent pathways in the vasculature. Accordingly we will perform our exercise protocol on global (Nrf2-/-, PGC-1-/-) and endothelial-specific loss-of-function models (ECKONrf2, ECKOPGC-1), and assess the pathways outlined above in Aim1. We will then determine if PGC-1 is sufficient to mimic exercise-induced vascular adaptation with an animal model of endothelial-specifc PGC-1 upregulation we have created that features enhanced NO bioactivity. Finally, we will determine the mechanisms regulating Nox4 and its contribution to the endothelial response to endurance exercise. Using an established carotid-to-jugular shunt system, we will model the extent to which increased flow mimics the changes in eNOS/NO, antithrombotic activity, and antioxidant activity seen with exercise. We will then test this model n Nox4-/- and ECKONox4 mice and determine the impact on NO bioactivity and the antithrombotic and antioxidant pathways listed above. We will then use human and murine endothelial cell models of Nox4, AMP kinase, Nrf2, and PGC-1 manipulation to determine the molecular mechanisms whereby Nox4 dictates the endothelial response to endurance exercise with regards to NO bioactivity, thrombosis resistance, and antioxidant upregulation. The experiments outlined above should provide us with a solid working knowledge of how Nox4 contributes to vascular homeostasis. These data will be a key element of determining how ROS can be adaptive in the vasculature and, importantly, how ROS positively regulate NO bioactivity and thromboresistance. With this information in hand, we should have the requisite insight to design therapies that modulate vascular ROS and better predict their impact on normal vascular physiology and also the pathophysiology of vascular disease.